Acetylcholine receptors in normal and denervated rat diaphragm muscle. II. Comparison of junctional and extrajunctional receptors.

Acetylcholine (ACh) receptors have been purified separately from normal rat diaphragm muscle (junctional receptors) and from extrajunctional regions of denervated diaphragm (extrajunctional receptors) in order to compare their properties. The toxin-receptor complexes of the two receptors were indistinguishable by gel filtration and by zone sedimentation in sucrose gradients, and showed identical precipitation curves with rabbit antiserum to the eel ACh receptor. Both toxin-receptor complexes bind concanavalin A and are therefore probably glycoproteins. Low concentrations of d-tubocuratine (dTC) were more effective in decreasing the rate of toxin binding to junctional than to extrajunctional receptors. The apparent dissociation constant for dTC binding to the junctional receptor was 4.5 X 10 minus 8 M, whereas the value for the extrajunctional receptor was 5.5 X 10 minus 7 M. When the complexes were analyzed by isoelectric focusing, the junctional complex focused at approximately 0.15 pH unit lower than the extrajunctional complex. This result was also found with crude preparations of receptor. We conclude that junctional and extrajunctional receptors are similar but distinct molecules. The properties of receptors present in neonatal diaphragm muscle were also examined and found to be similar to those of receptors in denervated muscle, as shown by dTC inhibition and isoelectric focusing.     

Subunit structure and peptide mapping of junctional and extrajunctional acetylcholine receptors from rat muscle.

We have purified the junctional acetylcholine receptor from normal rat skeletal muscle and compared its structure with that of the extrajunctional receptor from denervated muscle. The two receptors from leg muscle were distinguished by isoelectric focusing and by reaction with sera from patients with myasthenia gravis. The junctional form of the acetylcholine receptor was purified from normal leg muscle by affinity chromatography on concanavalin A/Sepharose and cobrotoxin/Sepharose followed by sucrose gradient centrifugation. Analysis of radioiodinated receptor by polyacrylamide gel electrophoresis in sodium dodecyl sulfate indicated that the subunit structure of the junctional receptor was similar to that previously determined for the extra-junctional form (Froehner, S. C., et al. (1977) J. Biol. Chem. 252, 8589-8596), with major polypeptides, whose apparent molecular weights in 9% polyacrylamide gels were 45 000 and 51 000. In addition, several minor polypeptides were found. When the two receptors were labeled with different isotopes of iodine and run together on a sodium dodecyl sulfate gel, the subunits of one receptor could not be resolved from those of the other. As seen earlier with the extrajunctional form, the affinity alkylating reagent [3H]MBTA labeled the 45 000- and 49 000-dalton polypeptides of the junctional receptor. Peptide mapping showed that the two MBTA binding subunits are structurally related, although they are unrelated to the other polypeptides, and that the 45 000- and 51 000-dalton polypeptides of the junctional receptor were indistinguishable from those of the extrajunctional receptor. In addition, peptide mapping of the four subunits of acetylcholine receptor isolated from Torpedo californica electric organ showed that these four polypeptides appear to be structurally unrelated.     

Regulation of formyl peptide receptor binding to rabbit neutrophil plasma membranes. Use of monovalent cations, guanine nucleotides, and bacterial toxins to discriminate among different states of the receptor

The regulation by monovalent cations, guanine nucleotides, and bacterial toxins of [3H]FMLP binding to rabbit neutrophil plasma membranes was studied by using dissociation techniques to identify regulatory effects on separate receptor states. Under conditions of low receptor occupancy (1 nM [3H]FMLP) and in both Na+ and K+ buffers, dissociation is heterogenous, displaying two distinct, statistically significant off rates. [3H]FMLP binding was enhanced by substituting other monovalent cations for Na+. In particular, enhanced binding in the presence of K+ relative to Na+ was caused by additional binding to both rapidly and slowly dissociating receptors. Three receptor dissociation rates, two of which appear to correspond to the two affinity states detected in equilibrium binding studies, were defined by specific GTP and pertussis toxin (PT) treatments. Neither GTP, nor PT or cholera toxins (CT) had an effect on the rate of dissociation of [3H]FMLP from the rapidly dissociating form of the receptor. Both 100 microM GTP and PT treatments increased the percentage of rapidly dissociating receptors, correspondingly decreasing the percentage of slowly dissociating receptors. The observed changes in the rapidly and slowly dissociating receptors after GTP, PT, and CT treatments were caused by an absolute decrease in the amount of binding to the slowly dissociating receptors. However, complete inhibition of slowly dissociating receptor binding by GTP, PT, or both was never observed. Both GTP and PT treatments, but not CT treatment, increased by two-fold the rate of dissociation of 1 nM [3H]FMLP from the slowly dissociating form of the receptor, resulting in a third dissociation rate. Thus, slowly dissociating receptors comprise two different receptor states, a G protein-associated guanine nucleotide and PT-sensitive state and a guanine nucleotide-insensitive state.     

Kinetic identification of a two-state glucagon receptor system in isolated hepatocytes. Interconversion of homogeneous receptors

A detailed kinetic study was performed to investigate the interaction of glucagon with receptors on freshly isolated hepatocytes. Competition binding assay results fit a mathematical expression for a single site noncooperative model of binding. Glucagon was shown to bind with first-order kinetics at six-hormone concentrations (0.02-0.50 nM) at 0 and 37 degrees C. The observed pseudo-first-order rate constants are directly proportional to the hormone concentration at 0 degree C, but display a downward deviation from linearity at 37 degrees C. Dissociation of glucagon exhibited biexponential character at 37 degrees C which was not seen at 0 degree C. The biphasic dissociation at 37 degrees C was resolved into rapid (t1/2 = 1.9 min) and slow (t1/2 = 27.7 min) components. The distribution of the total bound hormone between the rapidly and slowly dissociating complexes was not dependent upon the extent of receptor occupancy. The absolute quantity of rapidly dissociating hormone-receptor complexes was constant at all times examined; however, the fraction of slowly dissociating hormone-receptor complexes was found to increase with increasing incubation time. The results indicate that a homogeneous population of hepatic receptors undergoes a time-dependent, temperature-dependent conversion from one state to another in a two-stage sequential manner.     

A rapid ion-exchange assay for detergent-solubilized inositol 1,4,5-trisphosphate receptors.

We describe a rapid ion-exchange syringe assay for [3H]inositol 1,4,5-trisphosphate binding to detergent-solubilized receptors. In extracts of rat cerebellar membranes, the assay resolves rapidly dissociating ligand complexes, detecting two to three times higher receptor abundance than conventional gel filtration spun column assays, and provides evidence for two classes of IP3-binding sites, representing 0.5-1.0% of total cerebellar membrane protein. Receptors purified from bovine and rat cerebellum exhibit a single class of high-affinity sites, with equilibrium dissociation constants (Kd = 4-8 nM) reflecting 20 to 25-fold higher affinity than reported in studies with spun-column methods.     

Cholinergic sites in skeletal muscle. I. Denervation effects.

Cholinergic interactions in systems derived from rat skeletal mixed muscle are detailed. The isotherms of the binding of [125I]diiodo-alpha-bungarotoxin over the range of 10(-10)-10(-5) M toxin have been separated into a "nonspecific" component exclusive to the toxin and a "specific" component that binds both the toxin and d-tubocurarine. The "specific" component appears to reflect two independent sets of binding sites. One of the sets has an affinity constant on the order of 10(9) M-1. Following denervation, the number of sites in this high-affinity set begins to increase after 3 days, reaches a peak (28-fold higher than normal) on the 8th day, and begins to decline. Similar results are obtained when sensitivity of this set to an antibody derived from patients with myasthenia gravis is examined. This sensitivity is reflected by the inhibition of the alpha-bungarotoxin binding by the myasthenic IgG fraction. Following denervation, sensitivity first appears on day 3 progresses coincidentally with the increase in new sites in the set. The charcteristics of this set suggest that it represents the acetylcholine receptor and that the new sites appearing during the course of denervation are extrajunctional receptor sites. The interaction with the myasthenic IgG indicates an antigenic difference between junctional and extrajunctional receptors. The second set of specific binding sites has an affinity constant on the order of 10(5) M-1. The number of sites in this set increases only fivefold as a result of denervation. The increase also begins between days 2 and 3. The definition of this low affinity set of sites is not presently clear.     

Multiple Molecular Forms of Acetylcholine Receptors in Cultured Skeletal Muscle Cells: Subcellular Localization and Characterization

Abstract: Skeletal muscle cells of newborn rats, cultured in the absence of neuronal influence, were found to contain two types of cell surface acetylcholine receptors as demonstrated by isoelectric focusing. The isoelectric points of the two types of receptors were indistinguishable from those of junctional and extrajunctional types of receptors in mature animals. The cultured cells had two classes of intracellular α-bungarotoxin (αBT) binding components; one had the same sedimentation coefficient as that of surface receptors (9S), and the other had much smaller apparent molecular weights. Only a single major component was detected by isoelectric focusing analysis of the 9s intracellular aBT binding component, with a PI value close to that of the extra junctional receptor. These results suggest that the junctional and extrajunctional types of receptors may be synthesized through a common precursor.     

Binding of colchicine to purified microtubule protein.

The binding of colchicine to tubulin, purified by two cycles of assembly-disassembly, has been studied. Equilibrium studies indicated a dissociation constant which declined during incubation approaching a minimum value of approximately 0.30 times 10- minus 6 M after 13 hours of incubation. Because tubulin is unstable during prolonged incubation (t1/2 of 5.2 hours for free tubulin, t1/2 of 12.5 hours for tubulin bound to colchicine), the equilibrium Kd was felt to be an overestimation of the true Kd. The rate constant of dissociation (k-1 equal to 0.009 hour- minus 1 hour- minus 1) and the rate constant of association (k1 equal to 0.37 times 10-6 M-minus 1) were measured under conditions designed to circumvent or correct for tubulin instability. The dissociation constant determined by the ratio k-1/k1 was 0.024 times -minus 6 M. To determine whether the discrepancy between the "equilibrium" and "kinetic" determined dissociation constants could be accounted for on the basis of tubulin instability, the binding reaction was computer-simulated using the measured association and dissociation rate constants and the rate constants for decay of bound and free tubulin. Computer simulation was in close agreement with the experimentally determined behavior of the reaction during a 13-hour incubation. It is concluded that the Kd determined by equilibrium methodology results in a considerable overestimation due to the instability of tubulin, and that the best estimate for the Kd of the colchicine-tubulin equilibrium is the value determined by the ratio of the rate constants.     

Site-site interactions among insulin receptors. Characterization of the negative cooperativity.

By studying the dissociation of 125I-instulin from its receptors in the absence and phe negatively cooperative type for the insulin receptors. In the present study we extend oy purified mouse and rat liver membranes as well as in human circulating monocytes and human cultured lymphocytes demonstrated negative cooperativity that was extraordinarily simn membranes more slowly than it does from its receptors on whole cells. The dissociaty a small percentage of the receptor sites (1 to 5%), are sufficient to accelerate dissociation of hormone from receptor. At these insulin concentrations insulin is entirely monomeric, and in fact at higher concentrations of insulin (greater than 10(-7) M) where insulin dimers predominate, the cooperativity effect is progressively lost. The dissociation rate of 125I-insulin alone (that is at very low fractional saturation of receptors) was markedly accelerated by dripping the pH from 8.0 to 5.0, whereas the dissociation of 125I-insulin at high receptor occupancy was only slightly accelerated by the fall in pH. The dissociation rate was directly related to temperature, but the dissociation rate of 125I-insulin at low receptor occupancy was much more affected by reduction in temperature and showed a sharp transition at 21 degrees. Urea at concentrations as low as 1 M produced a marked acceleration of 125I-insulin dissociation. Divalent cations (calcium and magnesium) appear to stabilize the insulin-receptor interaction, since higher degrees of receptor occupancy were required to achieve a given rate of dissociation of 125I-insulin. These data make it likely that the insulin receptors exist as oligomeric structures or clusters in the plasma membrane. Insulin receptor sites appear to switch from a "slow dissociating" state to a "fast dissociating" state when their occupancy increases; the proportion of sites in each state is a function of occupancy of the receptor sites by the insulin monomer as well as of the physiochemical environment. Other models which could explain apparent negative cooperativity besides site-site interactions, i.e. polymerization of the hormone, steric or electrostatic hindrance due to ligand-ligand interactions, or unstirred (Noyes-Whitney) layers are considered unlikely in the case of insulin receptors on both experimental and theoretical grounds.     

Characteristics of Partially Purified Nerve Growth Factor Receptor

Receptors for the nerve growth factor protein (NGF) have been isolated from three cell types [embryonic chicken sensory neurons (dorsal root sensory ganglia; DRG), rat pheochromocytoma (PC12) and human neuroblastoma (LAN-1) cells] and have been shown to be similar with respect to equilibrium dissociation constants. The present results demonstrate that there are multiple molecular weight species for NGF receptors from DRG neurons and PC12 cells. NGF receptors can be isolated from DRG as four different molecular species of 228, 187, 125, and 112 kilodaltons, and PC12 cells as three molecular species of 203, 118, and 107 kilodaltons. The NGF receptors isolated from DRG show different pH-binding profiles for high- and low-affinity binding. High-affinity binding displays a bell-shaped pH profile with maximum binding between pH 7.0 and 7.9, whereas low-affinity binding is constant between pH 5.0 and 9.1, with a twofold greater binding at pH 3.6. At 22 degrees C, the association rate constant was found to be 9.5 +/- 1.0 X 10(6) M-1 s-1. Two dissociation rate constants were observed. The fast dissociating receptor has a dissociation rate constant of 3.0 +/- 1.5 X 10(-2) s-1, whereas the slow dissociating receptor constant was 2.4 +/- 1.0 X 10(-4) s-1. The equilibrium dissociation constants calculated from the ratio of dissociation to association rate constants are 2.5 X 109-11) M for the high-affinity receptor (type I) and 3.2 X 10(-9) M for the low-affinity receptor (type II). These values are the same as those determined by equilibrium experiments on the isolated receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of exposure to charcoal and ion-exchange chromatography on the dissociation rate of estrogen from the nuclear estrogen receptor of hen oviduct

The method of initiating dissociation of 3H-estradiol from the nuclear estrogen receptor of hen oviduct was found to have a profound effect on the dissociation rate. Likewise, prior exposure to charcoal or partial purification by ion-exchange chromatography had an effect on the dissociation rate. When the reaction was initiated by isotopic dilution with the addition of 1 microM unlabeled estradiol, dissociation of the complexes was rapid (t 1/2 approximately 3 min). When the reaction was initiated by the addition of charcoal to adsorb free steroid, the dissociation of the complexes proceeded slowly (t 1/2 approximately 30 min). Partial purification of the receptors by DEAE-Sephacel chromatography or 15 min exposure to charcoal at 0 degree C prior to initiation of the dissociation reaction by isotopic dilution produced a form of the receptor that exhibited an intermediate dissociation rate (t 1/2 approximately 10 min). The partially purified receptor that exhibited an intermediate dissociation rate was reconverted to the rapidly dissociating form in a reconstitution experiment. These data raise the possibility of a nuclear substance that regulates the rates of estrogen dissociation.     

Three states for the formyl peptide receptor on intact cells

Three distinct states of the formyl peptide receptor have been described. These are: 1) the ternary complex of ligand, receptor, and G protein (LRG); 2) the rapidly dissociating occupied receptor (ligand-receptor complex (LR]; and 3) a desensitized slowly dissociating guanine nucleotide-insensitive receptor (desensitized ligand-receptor complex ("LRX"]. During cell activation there is a rapid interconversion among receptor states from a rapidly dissociating form (t 1/2 approximately 10 s) to a slowly dissociating form (t 1/2 greater than or equal to 2 min). Neither the dynamics of the states nor their interconversion is influenced by ribosylation of G protein in the presence of pertussis toxin. In contrast to ribosylation, treatment of cells with either 2-deoxyglucose or fluoride ion, both of which lead to a loss of adenine and guanine nucleotides, causes a time-dependent change in ligand dissociability. After short periods of treatment (5-15 min) rapid dissociation is observed; after longer times (30-60 min), slow dissociation is once again detected. When intact cells are first ribosylated and then energy-depleted, only a rapidly dissociating receptor is detected. These results are discussed in terms of a model with the following elements: 1) intact cell dynamics during cell activation are dominated by an energy-dependent interconversion from LR to LRX; 2) under activation conditions, LRG appears and disappears too rapidly to be detected; 3) in cells depleted of energy and guanine nucleotide, LRG is stabilized; 4) in cells both ribosylated and depleted of energy, LR is stabilized.     

Interaction between the chemotactic cAMP receptor and a detergent-insoluble membrane residue of Dictyostelium discoideum. Modulation by guanine nucleotides

Cells from Dictyostelium discoideum carry chemotactic cAMP receptors on their surface. Kinetic studies have revealed the existence of two slowly dissociating, high affinity receptor forms (SS and S) and one or more fast dissociating, low affinity forms (F) (Van Haastert, P.J.M., and De Wit, R.J.W. (1984) J. Biol. Chem. 259, 13321-13328). We have studied the interaction of these different cAMP-receptor types with a detergent-insoluble membrane residue. Isolated D. discoideum membranes were extracted with the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS), which was previously shown to be the only detergent in the presence of which cAMP receptor binding is completely preserved (Janssens, P. M. W., and Van Driel, R. (1986) Biochim. Biophys. Acta 885, 91-101). The protein composition of the CHAPS-insoluble membrane residue appeared to be similar to that of the Triton X-100-insoluble membrane skeleton. Cyclic AMP binding studies revealed a specific association of the slowly dissociating cAMP receptors (SS and S forms) with this CHAPS-insoluble residue. All fast dissociating (F type) receptors were solubilized by CHAPS. GTP induced a transition of 75% of the SS and S receptors to faster dissociating forms. This transition was accompanied by the release of an equal number of receptors from the residue. These effects of GTP required that the cAMP receptor was occupied, and were completely reversible. After removal of the guanine nucleotide SS and S type receptors reappeared, bound to the residue, with a t1/2 of 5-10 min at 0 degrees C. We conclude that a detergent-insoluble membrane residue is involved in signal transduction via the chemotactic cAMP receptor. Both receptor occupation and a guanine nucleotide binding protein control receptor-residue interaction.     

Scintillation proximity assay as a high-throughput method to identify slowly dissociating nonpeptide ligand binding to the GnRH receptor

Many nonpeptide antagonists of the gonadotropin-releasing hormone (GnRH) receptor, as well as other drug targets, possess a broad range of dissociation kinetic rate constants. Current methods to accurately define kinetic rate parameters such as K(on) and K(off) are time and labor intensive, prompting the development of a screening assay to identify slowly dissociating compounds for follow-up rate constant determination. The authors measured inhibition binding constants (K(i)) for GnRH receptor antagonists after 30 min and 10 h of incubation and observed several compounds with markedly decreased K(i) values over time (Ki(30 min)/Ki(10 h) > 6). They used scintillation proximity assay technology to perform these binding experiments because this homogeneous assay does not have a fixed termination end point as does filtration binding, permitting successive readings to be taken from the same assay plate over an extended period of time. They also used a quantitative method of kinetic rate analysis to confirm that a large disparity between a compound's K(i) value at 30 min and 10 h could identify compounds that dissociate slowly. Thus, the K(i) ratio can be used to screen for and select compounds to test using more quantitative, albeit lower throughput methods to accurately define kinetic rate constants.     

Co-localization of nitric oxide synthase I (NOS I) and NMDA receptor subunit 1 (NMDAR-1) at the neuromuscular junction in rat and mouse skeletal muscle

Nitric oxide synthase I (NOS I) has been localized to the skeletal muscle sarcolemma in a variety of vertebrate species including man. It is particularly enriched at neuromuscular junctions. Recently, the N-methyl-d-aspartate (NMDA) receptor subunit 1 (NMDAR-1) has been detected in the postjunctional sarcolemma of rat diaphragm, providing a clue as to the possible source of Ca²⁺ ions that are necessary for NOS I activation. To address this possibility, we studied the distribution of NMDAR-1 and NOS I in mouse and rat skeletal muscles by immunohistochemistry and enzyme histochemistry. NMDAR-1 and NOS I were closely associated at neuromuscular junctions primarily of type II muscle fibers. NOS I was also present in the extrajunctional sarcolemma of this fiber type. Dystrophin, β-dystroglycan, α-sarcoglycan, and spectrin were found normally expressed in both the junctional and extrajunctional sarcolemma of both fiber types. By contrast, in the muscle sarcolemma of MDX mice, dystrophin and dystrophin-associated proteins were reduced or absent. NOS I immunoreactivity was lost from the extrajunctional sarcolemma and barely detectable in the junctional sarcolemma. NOS I activity was clearly demonstrable in the junctional sarcolemma by NADPH diaphorase histochemistry, especially when the two-step method was used. NMDAR-1 was not altered. These data suggest that different mechanisms act to attach NOS I to the junctional versus extrajunctional sarcolemma. It may further be postulated that NMDA receptors are involved not only in the regulation but also sarcolemmal targeting of NOS I at neuromuscular junctions of type II fibers. The evidence that glutamate may function as a messenger molecule at vertebrate neuromuscular junction is discussed.     

Degradation rate of acetylcholine receptors inserted into denervated vertebrate neuromuscular junctions

Many studies exist on the effect of denervation on the degradation of acetylcholine receptors (AChRs) at the vertebrate neuromuscular junction (nmj). These studies have described the behavior of either the total population of junctional receptors at different times after denervation, or of the receptors present at the time of denervation (referred to as original receptors). No experimental studies yet exist on the degradation rate of the receptors newly inserted into denervated junctions. In the previous studies, the original receptors of mouse sternomastoid muscles were found to retain the slow degradation (t 1/2) of approximately 8-10 d of innervated junctional receptors for up to 10 d after denervation before accelerating to a t 1/2 of approximately 3 d. The total junctional receptors, on the other hand, showed a progressive increase in degradation rate from a t 1/2 of 8-10 d to a t 1/2 of 1 d. To reconcile these earlier observations, the present study examines the degradation of new receptors inserted into the nmj after denervation. To avoid possible contamination of the data with postdenervation extrajunctional receptors, we used transmission electron microscope autoradiography to study only receptors located at the postjunctional fold of the nmj. We established that the new receptors inserted into denervated junctions have a t 1/2 of approximately 1 d, considerably faster than that of the original receptors and equivalent to that of postdenervation extrajunctional receptors. Both original and new receptors are interspersed at the top of the junctional folds. Thus, until all the original receptors are degraded, the postjunctional membrane contains two populations of AChRs that maintain a total steady-state site density but degrade at different rates. The progressive increase in turnover rate of total AChRs therefore reflects the combined rates of the original and new receptors, as earlier postulated by Levitt and Salpeter (1981).     

Alteration of binding properties and cytoskeletal attachment of nerve growth factor receptors in PC12 cells by wheat germ agglutinin

Incubation of PC12 cells preloaded with 125I-nerve growth factor (NGF) reveals rapidly and slowly dissociating binding components indicative of a heterogeneous population of receptors. If the cells are previously exposed to wheat germ agglutinin (WGA) for 30 min, NGF now binds to an apparently homogeneous receptor population which exhibit slow dissociation kinetics. Total binding is also reduced by 50%. If WGA is added subsequent to 125I-NGF, total binding is not diminished, but rapidly dissociating receptors occupied with NGF are all converted to the slowly dissociating form. This conversion of receptors occurs rapidly, reaching completion within 2 min at 37 degrees or 4 degrees C, and is unaffected by metabolic energy poisons, suggesting that WGA- induced slowly dissociating receptors are not the product of internalization. The effects of the lectin are blocked by the sugar N- acetyl-D-glucosamine, and the lectin-induced slowly dissociating receptors are converted back to rapidly dissociating receptors by addition of this same sugar. WGA also affects the association of the NGF receptor with the Triton X-100 cytoskeleton. Greater than 90% of bound 125I-NGF becomes associated with Triton X-100 insoluble cytoskeletons in the presence of the lectin, compared with less than 20% before lectin addition. Cytoskeleton association of the NGF receptor by WGA shows similar kinetics as the conversion of rapidly to slowly dissociating receptors. This interaction may be involved in the alteration of NGF-receptor binding properties produced by this lectin.     

Kinetic analysis of estrogen receptor homo- and heterodimerization in vitro

The coexistence of ERalpha and ERbeta suggests that active receptor complexes are present as homo- or heterodimers. In addition each of three forms of active receptors may trigger different cellular responses. A real-time biosensor based on surface plasmon resonance was used as instrument to determine binding kinetics of homo- and heterodimerization of estrogen receptor alpha and beta. Partially purified full-length estrogen receptor alpha was expressed intracellularly as a C-terminal fusion to a hexa-histidine tag using the baculovirus-expression system. Purified estrogen receptor alpha and beta without tags were used as partners in the dimerization process. An association rate constant of 3.6 x 10(3) to 1.5 x 10(4)M(-1)s(-1) for the homodimer formation of ERalpha and 5.7 x 10(3) to 1.5 x 10(4)M(-1)s(-1) for the heterodimer formation was found assuming a pseudo first-order reaction kinetic. The equilibrium dissociation constant for homodimerization of ERalpha was 2.2 x 10(-8) to 5.4 x 10(-8) and 1.8 x 10(-8) to 2.6 x 10(-8)M for the heterodimer formation. The homo- and heterodimer formation was characterized by a slow association kinetics and kinetic rate constants were within the same range.     

Regulation of ligand-receptor dynamics by guanine nucleotides. Real-time analysis of interconverting states for the neutrophil formyl peptide receptor

Intact neutrophils exhibit interconverting active and inactive receptor states with half-times for dissociation of 10 s and 2 min, respectively. We examined the effect of guanine nucleotides on ligand-receptor dynamics at 37 degrees C in neutrophils permeabilized with digitonin using continuous fluorometric measurements. The permeabilized cells exhibit a single class of slowly dissociating receptors with a half-time similar to the inactive state. The slowly dissociating state is lengthened in the presence of 10 mM by Mg2+ about two-fold but is relatively insensitive to substitutions of Na+ or K+. When guanine nucleotide is added the receptors dissociate uniformly with a half-time similar to the active state but are sensitive to the substitution of Na+ or K+ (K+ or K+/Mg2+ approximately 10 s; Na+ or Na+/Mg2+ approximately 4 s). When receptors in permeabilized cells are ADP-ribosylated with pertussis toxin the rapidly dissociating state is detected. In the presence of nonsaturating nucleotide or incomplete ribosylation, complex rates of ligand dissociation intermediate between the active and inactive forms are observed. Micromolar concentrations of Ca2+ block the effect of guanine nucleotide on the receptor. The relationships between ligand-receptor dynamics in intact neutrophils and interconverting states regulated by guanine nucleotides and ions in permeabilized cells are discussed.     

Study of the receptor for black widow spider neurotoxin. I. Characteristics of membrane-bound and solubilized receptors from the bovine brain

Iodine-125 labelled alpha-latrotoxin from the venom of Central Asia black widow spider Latrodectus mactans tredecimguttatus binds specifically to the bovine brain membrane receptor producing a stable slowly dissociating complex with Kd = 1.6 x 10(-10) M and Bmax = 0.5 pmol/mg protein. Treatment of the complex with alkaline high-salt buffer induces reversible dissociation of the bound toxin. The antitoxin polyclonal antibody does not increase the dissociation rate of the bound toxin. Wheat germ lectin as well as concanavalin A inhibit the toxin binding to the membrane receptor. The receptor is solubilized with ionic and non-ionic detergents, and methods of latrotoxin binding assay are developed. The solubilized receptor is shown to retain high affinity to toxin, its binding activity being stable but critically dependent on the presence of calcium ions. Chromatographic properties of the receptor suggest its glycoprotein nature.